This proposal is intended for support by the Biophysics and Physiological Sciences (BPS) Program, Biophysics Section of NIGMS, which has the explicit mission to "encourage the development and improvement of instruments, devices, and methodologies for biomedical research." The proposal contains five projects that are concerned with pulse (or time-domain) saturation-recovery electron-spin resonance (ESR) instrumentation and methodology. Biomedical applications of this technique receive minimal explicit attention in the proposal because of the 20 page limit. However, more than half of the 35 papers written in the previous 5-year funding period are in the category of "applications, " and this aspect of the work will continue. The five projects are: 1) Adaptation of two commercial digital signal processors to the experiment; one with very high digitization rate (500 MHz), but lower overall signal processing capability, and one with very high signal processing capability (31,200 acquisitions per sec of 512 points each), but lower digitization rate. 2) Introduction of a new two-dimensional saturation-recovery pulse sequence that will improve our ability to deconvolve multiple exponentials and thereby to study translational diffusion, particularly in membranes. 3) Continued development of a unique capability of this laboratory: multi-microwave frequency saturation-recovery (2-4, 10 and 19 GHz). Project 3 also initiates a new line of research: multifrequency studies of free-induction decays. 4) Development of numerous loop-gap resonators for improved sensitivity and reduced deadtime. 5) Study of the use of multiquantum ESR as a technique for detecting saturation-recovery signals. Saturation-recovery ESR yields significant information concerning translational diffusion coefficients of biomolecules, transport and consumption of oxygen by respiring cells, distances between two labeled sites in proteins, and the nature of rotational motions, particularly of proteins in membranes. A recent and significant application, one that involves site-specific mutagenesis, yields structural information on membrane proteins. The two digital signal processors, Analytek series 2000 and DSP Technology 2101, will require some additional engineering development, but this development is not extensive. The costs of the units are relatively modest, and the designs will be readily transportable to other laboratories, which should increase the overall use of ESR saturation-recovery methodology.